Organisms from bacteria to humans use a circadian clock to control daily biochemical, physiological, and behavioral rhythms. This clock affects human physiology, and disruptions of normal clock function can cause mental health problems, including manic depression. From studies of Neurospora and Drosophila we are beginning to understand the molecular mechanism of the clock. However, the way in which the clock regulates gene expression is largely unknown. We have identified four classes of Neurospora clock-controlled genes whose mRNA levels peak at dawn, noon, dusk and midnight. The clock in humans also regulates genes whose expression peaks at different times. Thus, a critical question is how the clock signals genes to be expressed at different times of day. One hypothesis is that a single output pathway from the clock incorporates time delays to trigger sequential expression of the different gene sets. A second hypothesis is that there are time-of-day-specific output pathways from the clock, with each pathway regulating one of the gene classes. We have developed a genetic selection to identify output-pathway genes and have found four genes (cop-1, cop-2, cop-3 and nrc-2 (cop-5)) required for rhythmicity of one or more clock-controlled genes. To test the two hypotheses, we will clone the four genes and determine how the clock affects expression, localization and/or modification of their mRNA and protein products. To map the output pathways, we will determine how mutations in any one of the four genes affects the expression of the different classes of clock-regulated genes, and the expression level, localization and/or modification of the other cop genes and COP proteins. Using biochemical and genetic methods, we will determine if known clock components interact with the COP proteins to signal time-of-day information to downstream genes. Our studies will have a significant impact on understanding how circadian clocks control overt rhythmicity and will potentially provide novel approaches for therapies for human diseases that result from circadian dysfunction.